1. Field of the Invention
This invention relates to melanocortin receptors from mammalian species and the genes corresponding to such receptors. Specifically, the invention relates to the use of mammalian melanocortin receptors for the development of naturally-occurring and synthetic agonists and antagonists specific for a mammalian melanocortin receptor, and the use of such agonists and antagonists for treatment and alleviation of dysfunction and disease. Specifically, the invention relates to development of naturally-occurring and synthetic agonists and antagonists specific for a mammalian melanocortin receptor termed MC5-R (see U.S. Pat. No.5,622,860, incorporated by reference). Such naturally-occurring and synthetic agonists and antagonists specific for the MC5-R receptor are provided for the treatment, control, amelioration and alleviation of diseases, and dysfunctional and abnormal states related to thermoregulatory disorders, as well as other diseases relating to exocrine gland disorders, including lacrimal gland dysfunction and sebaceous gland disorders including acne and other skin problems. Also provided by the invention are nucleic acids, constructs, vectors and methods for producing an animal having homozygous disruption of both endogenous MC5-R melanocortin receptors, preferably a rodent and most preferably a mouse. Such rodents, termed "gene knockout"rodents in the art, are also advantageously provided.
2. Background of the Invention
The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, .alpha.-melanocyte stimulating hormone (.alpha.MSH), and adrenocorticotropic hormone (ACTH) have well-understood roles in control of melanocyte and adrenocortical function, respectively. Both of these hormones are also found in a variety of forms with unknown functions, for example, .gamma.-melanocyte stimulating hormone (.gamma.MSH), which has little or no ability to stimulate pigmentation (Ling et al., 1979, Life Sci. 25:1773-1780; Slominski et al., 1992, Life Sci. 50:1103-1108). A melanocortin receptor gene specific for each of the .alpha.MSH, ACTH and .gamma.MSH hormones has been discovered by some of the present inventors (see U.S. Pat. Nos. 5,280,112 and 5,532,347 and U.S. application Ser. No. 08/044,812, incorporated by reference herein). In addition, two other melanocortin receptor genes have been discovered by some of the present inventors (see Lu et al, 1994, Nature 371:799-802, Mountjoy et al., 1994, Molec. Endocrinol. 8:1298-1308) and others (see U.S. Pat. No. 5,622,860; Gantz et al., 1993, J Biol. Chem. 268:15174-15179 and Labbe et al., 1994, Biochem. 33:4543-4549). Thus far, the biological activities of the melanocortin peptides appear to be mediated by a family of five G protein coupled receptors (see Cone, 1996 for a review).
Along with the well-recognized activities of .alpha.MSH in melanocytes and ACTH in adrenal and pituitary glands, the melanocortin peptides also have a diverse array of biological activities in other tissues, including the brain and immune system, and bind to specific receptors in these tissues with a distinct pharmacology (see Hanneman et al., in Peptide Hormone as Prohormones, G. Martinez, ed. (Ellis Horwood Ltd.: Chichester, UK) pp. 53-82; DeWied & Jolles, 1982, Physiol. Rev. 62:976-1059 for reviews). For example, POMC neurons are present in only two regions of the brain, the arcuate nucleus of the hypothalamus, and the nucleus of the solitary tract of the brain stem. Neurons from both sites project to a number of hypothalamic nuclei, including the paraventricular nucleus, lateral hypothalamic area, and ventromedial hypothalamic nucleus. A complete understanding of these peptides and their diverse biological activities requires the isolation and characterization of their corresponding receptors. Some biochemical studies have been reported in the prior art.
Shimuze, 1985, Yale J. Biol. Med. 58:561-570 discusses the physiology of melanocyte stimulating hormone.
Tatro & Reichlin, 1987, Endocrinology 121:1900-1907 disclose that MSH receptors are widely distributed in rodent tissues.
Sola et al., 1989, J Biol. Chem. 264:14277-14280 disclose the molecular weight characterization of mouse and human MSH receptors linked to radioactively and photoaffinity labeled MSH analogues.
Siegrist et al., 1991, J Receptor Res. 11:323-331 disclose the quantification of receptors on mouse melanoma tissue by receptor autoradiography.
Cone & Mountjoy, U.S. Pat. No. 5,532,347, issued Jul. 2, 1996, disclose the isolation of human and mouse .alpha.-MSH receptor genes and uses thereof (incorporated herein by reference).
Cone & Mountjoy, U.S. Pat. No. 5,280,112, issued Jan. 18, 1994, disclose the isolation of human and bovine ACTH receptor genes and uses thereof (incorporated herein by reference).
Mountjoy et al., 1992, Science 257:1248-1251 disclose the isolation of cDNAs encoding mammalian ACTH and MSH receptor proteins.
Cone et al., U.S. Ser. No. 08/044,812, filed Apr. 8, 1993, disclose the isolation of rat .gamma.-MSH receptor genes and uses thereof (incorporated herein by reference).
The distribution of expression of the known melanocortin receptors has largely fit expectations regarding the known biological activities of the melanocortin peptide ligands encoded by the POMC gene. The MC1-R, or classical MSH receptor, is expressed almost exclusively in melanocytes (Chhajlani and Wikberg, 1992, FEBS Lett. 309:417-420; Mountjoy et al., 1992, ibid.), where it regulates melanin synthesis. The MC2-R, or classical ACTH receptor, is expressed primarily in the adrenal cortex (Mountjoy et al., 1992, ibid.), where it regulates adrenocortical steroidogenesis (although this receptor is also expressed in adipocytes, explaining the ability of ACTH to stimulate lipolysis). The MC3-R and MC4-R are expressed mainly in the central nervous system in regions that are well-correlated with presumptive terminal fields originating from the two groups of POMC cell bodies in the arcuate nucleus of the hypothalamus and the nucleus of the solitary tract of the brainstem (Mountjoy et al., 1994, ibid.; Roselli-Rehfuss et al., 1993, Proc. Natl. Acad. Sci. USA 90:8856-8860). Recently, it has been shown that MC3-R and MC4-R regulate feeding behavior and metabolism (Fan et al., 1997, Nature 385:165-168; Huszar et al., 1997, Cell 88:131-141), grooming behavior (Adan et al., 1994), body temperatures (Tatro et al., 1990, Cancer Res. 50:1237-1242), and cardiovascular tone (Li et al., 1996, J. Neurosci. 16:5182-5188); see also U.S. patent application Ser. No. 08/706,281, filed Sep. 4, 1996 and incorporated by reference herein.
Numerous peripheral effects of POMC peptides have been reported. For example, removal of the neurointermediate lobe of the pituitary (which produces the POMC peptides) was demonstrated to decrease sebaceous lipid production (Thody and Shuster, 1973, Nature 245:207-209). The reduction was fully restored by concomitant A-MSH and androgen administration (Ebling et al., 1975, J. Endocrinol. 66:407-412). The lipid content of the preputial gland (a specialized sebaceous gland implicated in pheromone production in rodents; Bronson and Caroom, 1971, J. Reprod. Fertil. 25:279-282; Chipman and Alberecht, 1974, J. Reprod. Fertil. 38:91-96; Orsulak and Gawienowski, 1972, Biol. Reproduc. 6:219-223) has been shown to be stimulated by .alpha.-MSH. Injection of .alpha.-MSH has been shown to elicit several behavioral changes in the conspecific animals, including altered sexual attraction in male rats (Thody and Wilson, 1983, Physiol. Behav. 31:67-72), and modified aggression in male mice due to olfactory cues presumably from the preputial gland (Nowell et al., 1980, Physiol. Behav. 24:5-9). High affinity ACTH and MSH binding sites have also been reported to regulate lipolysis in adipocytes (Oelofsen and Ramachandran, 1983, Arch. Biochem. Biophys. 225:414-421; Ramachandran et al., 1976, Biochim. Biophys. Acta 428:339-346) and protein secretion in the lacrimal gland (Jahn, 1982, Eur. J. Biochem. 126:623-629; Tatro and Reichlin, 1987, ibid.).
The systemic effects of pituitary-derived peptides have been attributed to ACTH-mediated adrenocortical glucocorticoid production. The primary role of serum-derived ACTH is the regulation of adrenocortical glucocorticoid production. In response to physical or psychological stress, hypothalamic corticotropin releasing hormone stimulates the production of ACTH by anterior pituitary cells. Serum ACTH is elevated 3-5 fold, producing a subsequent 10-100 fold elevation in circulating cortisol or corticosterone. Glucocorticoids then support the response to stress, serving to stimulate hepatic gluconeogenesis and elevate blood glucose, and mobilize amino acid stores from muscle and fatty acids from adipose tissue. Glucocorticoids also have an important role in the resolution of immune responses, acting on numerous cell types to reduce inflammation.
One of the melanocortin receptors, termed MC-5, has been found by the present inventors to be widely-distributed in peripheral tissues, raising the possibility of non-steroidally mediated systemic effects of MSH/ACTH peptides. This receptor has been cloned from human, mouse, rat, and sheep, and is highly conserved, being approximately 80% identical amongst the mammals. Furthermore, the MC5-R is highly responsive to both .alpha.-MSH and ACTH, as determined by EC.sub.50 values for elevation of intracellular cAMP or activation of adenylate cyclase. Further investigation by the present inventors has demonstrated high levels of MC5-R gene expression in multiple exocrine tissues, including the Harderian, preputial, lacrimal, and sebaceous glands in rodents. The MC5-R has also been shown to be required for the production of porphyrins by the Harderian gland, and physiological concentrations of ACTH were demonstrated to regulate protein secretion by the lacrimal gland via binding to MC5-R.
The present inventors have now produced a mouse by targeted disruption of the MC5-R gene with a severe defect in water repulsion and thermoregulation due to decreased production of sebaceous lipids. Analysis of these mice revealed a requirement for MC5-R gene expression in multiple exocrine glands in vivo for the production of a diverse set of products, including lipids, proteins, and porphyrins, and suggested the existence of a coordinated system for the regulation of exocrine gland function by melanocortin peptides, related to thermoregulatory homeostasis, tear production and the production of skin and hair oils. Thus, these results produced for the first time in the art a need for the development of MC5-R receptor agonists and antagonists for the regulation of such biological processes and for the alleviation of diseases, dysfunctions and abnormal conditions related to exocrine gland function.